Clone： 5C11Other Names： BP50, TNFRSF5Isotype： Mouse IgG1, κ Description： CD40 is a 48 kD type I glycoprotein also known as BP50. It is a member of the TNFR superfamily primarily expressed on B cells, macrophages, follicular dendritic cells, endothelial cells, fibroblasts, and at low levels on plasma cells. CD40 has been reported to be involved in B cell differentiation, costimulation, isotype class-switching, and protection of B cells from apoptosis. Additionally, CD40 is important for T cell-B cell interactions. The ligand of CD40 is CD154 (CD40 ligand). The 5C3 antibody has been reported to promote B cell proliferation in the presence of anti-IgM, IL-4 or PMA, partially blocking CD40 binding to CD40L, and B cells rescue from apoptosis. Antibody Type： MonoclonalHost Species： MouseImmunogen： Human CD40 Recombinant ProteinFormulation： Antibodies are supplied in buffer containing stabilizer and 0.05% sodium azidePreparation： The antibody was purified by affinity chromatography.Storage & Handling： The antibody solution should be stored undiluted between 2°C and 8°C.Recommended Usage： 0.5ug/TExcitation Laser： Blue Laser (488 nm) Green Laser (532 nm)/Yellow-Green Laser (561 nm) LWB Cell Preparation： 1. For each patient sample, label three tubes A, B, and C. Label each tube with the sample identification number. 2. Place 50 µL PBS reagent into Tube A, Mouse IgG1 Control reagent into Tube B, and 0.5 µg of theCD40reagent into Tube C. 3. For each sample tube, use a fresh micropipettor tip and carefully add 5*105 pbmc into the bottom of each of the three labeled tubes. All tubes refill to 100ul. 4. Vortex thoroughly at low speed for 3 seconds and incubate for 15minutes at 4℃. Then repeat this step again. NOTE Protect samples from direct light during this incubation procedure . 5. After incubation, add 1 mL of PBS with 1% FBS to each tube. Centrifuge tubes at 1500rpm for 5 minutes at room temperature (20°C–25°C). 6. Aspirate the supernatant leaving approximately 50 µL of residual fluid in the tube to avoid disturbing the pellet. 7. Repeat 5,6 steps twice. 8. The secondary antibody used was Beckman Goat F（ab')2 Fragment Anti mouse IgG(H+L)-PE (BECKMAN COURTER IM0855) secondary antibody at 1/1000 dilution. 9. Place 100 µL secondary antibody into Tube B,C. Vortex thoroughly at low speed for 3 seconds and incubate for 15minutes at 4℃. Then repeat this step again. 10. Repeat 5,6 steps twice. 11. Add 0.3 mL of PBS to each tube and then vrtex thoroughly at low speed to resuspend the cell pellet in the residual fluid . Vortex thoroughly at low speed for 3 seconds. Make sure the cells are well mixed with the fixing solution. 12. The cells are now ready to be analyzed on the flow cytometer. Cap or cover the prepared tubes and store at 2°C– 8°C in the dark until flow cytometric analysis. NOTE Analyze the fixed cells within 2 hours after staining.Over 2 hours add 0.3 mLof PBS with 1% paraformaldehyde to each tube. For research use only. Not for diagnostic use. Not for resale. Suzhou Bright Scistar Biotech Co. Ltd will not be held responsible for patent infringement or other violations that may occur with the use of our products.
